This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and at increased risk for breast cancer among Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) A comprehensive literature review of the molecular changes in normal breast tissue at either normal risk or at high risk for breast cancer to define the molecular changes in early breast carcinogenesis and to guide the molecular characterization of breast epithelial cells collected from women at normal risk or at high risk for breast cancer under protocol 02-C-0077. c.) A comprehensive literature review of molecular changes contributing to the disparities in development, presentation and outcomes of breast cancer between Caucasian, Hispanic, and African American women. A comprehensive review of the literature has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN Breast Cancer Research, 15:208-220, 2013). This model identified multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. This model also emphasizes the need for defining the molecular characteristics of early carcinogenesis in these ethnic groups. Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of Caucasian, African American, and Hispanic women at normal risk and at increased risk for breast cancer. One hundred thirty women have been studied, 60 high risk subjects and 70 subjects at normal risk. The ductal architectural characteristics of breasts have been defined and correlated with the presence of ductal epithelial cell atypia. A significantly improved method of ductal epithelial cell sampling has been developed which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. These important findings were recently published (Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015). Extraction of a single intact ductal lavage sample (fluid and epithelial cells) or the separate frozen cellular component provided DNA and RNA suitable for multiple downstream molecular studies including RT-PCR of miRNA species, qPCR of the telomerase gene, whole genome DNA amplification, arrayCGH analysis, and microarray gene expression profiling. This method significantly expands our ability to define the molecular characteristics according to risk of breast ductal epithelium in women at different risks for breast cancer, and also introduces a much needed method for collecting ductal epithelial cells from women at normal risk for breast cancer, a critical control group for defining the multiple molecular characteristics of women at increased risk for breast cancer. Importantly,50% - 70% of women in the U.S. who develop breast cancer have no identifiable risk factors, and thus our findings should permit us to further characterize and identify at-risk women in this group, a major population of women in the U.S. Molecular studies to define numerical and structural chromosome abnormalities and gene and microRNA expression of normal at-risk breast epithelial cells from Caucasian, Hispanic and African American women are in progress. The second comprehensive literature review conducted as part of this project has identified important early changes of breast carcinogenesis in normal at-risk breast epithelium, including loss of heterozygosity/allelic imbalance resulting from small segmental deletions, DNA methylation of critical tumor suppressor genes including APC, RASSF1A, p16INK4A, BRCA1, and telomere shortening. Progression of each of these types of genomic abnormalities and the development of aneuploidy occurs in normal breast tissue at high risk for breast cancer. We have conducted preliminary studies of breast ductal epithelial cells by gene expression profiling from women at high risk compared with women at normal risk for breast cancer using the Affymetrix platform, and principal component analysis indicates distinct expression populations for these two groups and a striking differential expression of genes in the high risk group consistent with progression of breast carcinogenesis in these cells. To further define the molecular characteristics associated with increased risk for breast cancer we have collected under protocol 02-C-0077 breast epithelial cells from women at increased risk from two additional important risk groups a.) women at increased risk because of hormonal and anthropomorphic factors (nulliparity, late age of first childbirth, obesity [RR = 1.5 - 2.0), and b.) women at increased risk because of the presence of atypical ductal epithelial cells in their ductal lavage specimens (RR = 2.0 - 4.0). Identification of the molecular characteristics associated with these two categories of risk factors, combined with our studies of normal risk and of high risk epithelium, should provide critical information toward our objective of defining the breast carcinogenic pathway and for development of a molecular signature for risk assessment. Breast ductal fluid and ductal epithelial cells are being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid collected from these subjects, suggesting an important mechanism for the expansion of the cancerized field and risk within the breast and enhancement of progression through the carcinogenic pathway. We are also developing in this project a potentially very valuable resource, the development of normal breast epithelial cell lines from the ductal lavage samples from women at different risks for breast cancer. Demographic data including detailed risk assessment information is available for each subject. We are developing these cell lines in conjunction with Lonza Corp (Walkersville, MD). The development of a panel of cell lines according to different risks for breast cancer will provide abundant material for molecular profiling of risk characteristics, facilitate analysis of metabolic properties including response to exogenous mitogens and inhibitory substances, promote identification of alterations in signaling pathways according to risk, and allow development of an in vitro model useful for evaluation of chemoprevention drugs. The identification of specific abnormalities between Caucasian and African American breast cancer will also provide important correlative information for analysis of normal and high risk breast epithelium, and facilitate characterization of carcinogenic changes, potentially ethnic-specific, in at-risk breast epithelium.